Hepatitis C virus (HCV) has a positive-sense single-strand RNA genome and is a member of the genus Hepacivirus within the Flaviviridae family of viruses (Rice, 1996). As for all positive-stranded RNA viruses, the genome of HCV functions as mRNA, from which all of the viral proteins necessary for propagation are translated.
The viral genome of HCV is approximately 9600 nucleotides (nts) in length and consists of a highly conserved 5′ untranslated region (UTR), a single long open reading frame (ORF) of approximately 9,000 nts and a complex 3′ UTR. The 5′ UTR contains an internal ribosomal entry site.
The 3′ UTR consists of a short variable region, a polypyrimidine tract of variable length and, at the 3′ end, a highly conserved region of approximately 100 nucleotides. The last 46 nucleotides of this conserved region were predicted to form a stable stem-loop structure thought to be critical for viral replication.
The ORF encodes a large polypeptide precursor that is cleaved into at least 10 proteins by host and viral proteinases. These proteins are the structural proteins Core, E1, E2; p7; and the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, NS5B. The predicted envelope proteins contain several conserved N-linked glycosylation sites and cysteine residues. The NS3 gene encodes a serine protease and an RNA helicase and the NS5B gene encodes an RNA-dependent RNA polymerase.
A remarkable characteristic of HCV is its genetic heterogeneity, which is manifested throughout the genome. The most heterogeneous regions of the genome are found in the envelope genes, in particular the hypervariable region 1 (HVR1) at the N-terminus of E2. HCV circulates as a quasispecies of closely related genomes in an infected individual. Globally, seven major HCV genotypes (genotypes 1-6) and multiple subtypes (a, b, c, etc.) have been identified.
The nucleotide and deduced amino acid sequences among isolates within a quasispecies generally differ by 1-2%; those of different strains/isolates differ by 2-10%, whereas isolates of different subtypes and genotypes usually vary by>20% and>30%, respectively. Genotypes 1, 2 and 3 are found worldwide and constitute more than 90% of the HCV infections in North and South America, Europe, Russia, China, Japan and Australia. Throughout these regions genotype 1 accounts for the majority of HCV infections but genotypes 2 and 3 each account for significant percentage of infections.
More than 80% of individuals infected with HCV become chronically infected and these chronically infected individuals have a relatively high risk of developing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The only currently licensed therapy for chronic hepatitis C, interferon-alfa2 (IFN) in combination with ribavirin, induces a sustained viral response in less than 50-80% of treated patients depending on genotype. Consequently, HCV is currently the most common cause of end stage liver failure and the reason for about 30% of liver transplants performed in the U.S.
In addition, a number of recent studies suggested that the severity of liver disease and the outcome of therapy may be genotype-dependent. In particular, these studies suggested that infection with HCV genotype 1b and 3a were associated with more severe liver disease and that HCV genotype 1a and 1b might be associated with a poorer response to IFN therapy. As a result of the inability to develop a universally effective therapy against HCV infection, it is estimated that there are still more than 40,000 new infections yearly in the U.S. and about 3 million worldwide. Moreover, since there is no vaccine for HCV and as mentioned no effective treatment, HCV remains a serious public health problem.
Despite the intense interest in the development of vaccines and therapies for HCV, progress has been hindered by the absence of a useful cell culture system and the lack of convenient small animal models for laboratory study. For example, while replication of HCV in several cell lines has been reported, such observations have turned out not to be highly reproducible. And as described below only the JFH1 starin of HCV genotype 2a can grow in culture. The chimpanzee is the only HCV pathogenesis animal model. Consequently, HCV has been studied mainly by using clinical materials obtained from patients or experimentally infected chimpanzees, an animal model whose availability is very limited.
However, several researchers have recently reported the construction of infectious cDNA clones of HCV, the identification of which would permit a more effective search for susceptible cell lines and facilitate molecular analysis of the viral genes and their function.
Kolykhalov et al., (1997) and Yanagi et al. (1997, 1998) reported the derivation from HCV strains H77 (genotype 1a) and HC-J4 (genotype 1b) of cDNA clones of HCV that are infectious for chimpanzees. Subsequently, several other cDNA clones of genotype 1a (strains HCV-1 and TN), 1b (strains Con1 and HCV-N) and 2a (strains J6 and JFH1) were developed. However, while these infectious clones will aid in studying HCV replication and pathogenesis and will provide an important tool for development of in vitro replication and propagation systems, it is important to have infectious clones of all major HCV genotypes, given the extensive genetic heterogeneity of HCV and the potential impact of such heterogeneity on the development of effective therapies and vaccines for HCV.
In addition, synthetic chimeric viruses can be used to map the functional regions of viruses with different phenotypes. In flaviviruses and pestiviruses, infectious chimeric viruses have been successfully engineered to express different functional units of related viruses and in some cases it has been possible to make chimeras between non-related or distantly related viruses. For instance, the IRES element of poliovirus or bovine viral diarrhea virus has been replaced with IRES sequences from HCV.
The construction of an infectious chimera of two closely related HCV subtypes has been reported. The chimera contained the complete ORF of a genotype 1b strain but had the 5′ and 3′ termini of a genotype 1a strain (Yanagi et al., 1998).
Recently, it was shown, that transfection of RNA transcripts from cDNA clone of genotype 2a isolate JFH1 into Huh7 hepatoma cells led to productive infection of these cells with JFH1 virus (Wakita 2005, Zhong 2005). It is not known, why JFH1 can grow in cell culture and other HCV isolates cannot. To exploit the exceptional growth characteristics of JFH1 in cell culture, the construction of JFH1-based intra- and intergenotypic recombinants became a research focus. Thus, intragenotypic and intergenotypic recombinants have been constructed containing non structural proteins NS3-NS5B of genotype 2a isolate JFH1 and Core, E1, E2, p7, and NS2 from genotype 1a (strain H77 and TN), 1b (strain J4 and Con-1), 2a (strain J6), 2b (strain J8), 3a (strain S52, DBN, and 452), 4a (strain ED43), 5a (strain SA13), 6a (strain HK6a), and 7a (strain QC69). Transfection of RNA transcripts of cDNA clones of these recombinants led to productive infection of Huh7.5 human hepatoma cells (Pietschmann 2006, Gottwein 2007, Scheel 2008, Jensen 2008, Gottwein, 2009). However, for most of the intergenotypic recombinants, viability in Huh7.5 cells required acquisition of cell culture adaptive mutations, possibly enabling interaction of proteins of different genotype isolates. J6/JFH1 has also been found to be viable in chimpanzees and in the SCID-uPA mouse model (Lindenbach 2005, Lindenbach 2006).